Peptides derived from cadherin and methods of use thereof

ABSTRACT

The present invention provides polypeptides and peptides derived from cadherin. The polypeptides and peptides are useful in a method of inhibiting amyloid deposition and a method of inhibiting tumor metastasis. A method of determining susceptibility to Alzheimer&#39;s disease and a method of screening for agents that modify cadherin processing are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Application Ser. No.60/372,617, filed Apr. 11, 2002, the disclosure of which is incorporatedherein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Nos.AG-08200, AG-05138 and AG-17926 awarded by the National Institutes ofHealth. The government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

Classic cadherins, including epithelial (E)- and neural (N)-cadherins,are major cell-cell adhesion receptors involved in the development,maintenance and function of most tissues, including the nervous system,epithelia and endothelia. In addition, cadherins play important roles incell signaling, proliferation and differentiation. In cadherin-basedadherens junctions (CAJ), the extracellular domains of transmembranecadherins promote cell-cell adhesion by engaging in Ca⁺⁺-dependenthomophilic interactions, while the cytoplasmic domains are linked to theactin cytoskeleton via α- and β-catenins. Post-translational regulationof cadherin adhesive activities, including proteolytic processing ofcadherins and disassembly of CAJ, play crucial roles in rapid changes incell adhesion, signaling and apoptosis, but the molecular mechanismsinvolved in cadherin processing and CAJ disassembly remain mostlyunknown.

Presenilin-1 (PS1) is a polytopic transmembrane protein involved in mostcases of early-onset familial Alzheimer's disease (FAD). Cellular PS1 iscleaved to yield an N-terminal (PS1/NTF) and a C-terminal (PS1/CTF)fragment. Following cleavage, the resultant PS1 fragments form a stable1:1 heterodimer which binds to the cytoplasmic juxtamembrane region ofE-cadherin (Baki et al., (2001) Proc. Natl. Acad. Sci. USA,98:2381-2386). PS1 is found in the ER-Golgi system, but upon formationof cell-cell contacts PS1 concentrates at intercellular sites at thecell surface where it forms complexes with the CAJ. In addition toE-cadherin, PS1 forms complexes with N-cadherin and it has beenlocalized at synaptic sites. Recently it was reported that PS1 regulatesa γ-secretase cleavage of both APP and Notch receptor and stimulatesAβ-production (Herreman et al., (2000) Nat. Cell Biol. 2:461-2).

In the brain, PS1 forms complexes with N-cadherin (Georgakopoulos et al.(1999) Mol. Cell. 4: 893-902), a type I transmembrane protein and amember of the classic cadherin family of Ca⁺⁺-dependent cell adhesionfactors (Gumbiner (1996) Cell 84: 345-357). Both proteins are expressedin neurons and have been found at the synapse (Georgakopoulos et al.(1999); Uchida et al. (1996) J. Cell. Biol. 135: 767-779). N-cadherinhomophilic interactions are thought to play an important role in holdingtogether pre- and post-synaptic membranes (Fannon and Colman (1996)Neuron 17: 423-434) and N-cadherin has been shown to undergo molecularchanges in response to synaptic activity (Tanaka et al. (2000) Neuron25: 93-107). Furthermore, N-cadherin promotes axonal outgrowth andregulates synaptogenesis and long term potentiation (LTP) (Goda (2002)Neuron 35: 1-3).

PS1 is important for the γ-secretase cleavages of the amyloid precursorprotein (APP), which result in the production of the Aβ peptide ofAlzheimer's disease (AD) (De Strooper et al. (1998) Nature 391:387-390). In addition to the classic γ-secretase cleavages of APPdefined by the C-terminus of various Aβ species, the PS1/γ-secretasesystem promotes the γ-secretase-like, or ε-cleavage (Weidemann et al.(2002) Biochemistry 41: 2825-2835) of several type I transmembraneproteins, including APP, Notch1 receptor, E-cadherin and CD44. Althoughthis cleavage is also sensitive to γ-secretase inhibitors, it takesplace further downstream from the amyloidogenic γ-secretase cleavages ata site closer to the membrane/cytoplasm interface than the γ-cleavages(De Strooper et al. (1999) Nature 398: 518-522). It has been discoveredin accordance with the present invention that in certain cases, likeE-cadherin (see FIG. 1), the ε-cleavage is greatly stimulated by calciumimbalance or apoptosis. The ε-cleavage results in the release of solublecytosolic peptides containing the intracellular domains (ICDs) of thecleaved substrate proteins. Some of these peptides have been shown tomigrate to the nucleus where they may act as regulators of geneexpression (for reviews see Ebinu and Yankner (2002) Neuron 34: 499-502;Fortini (2002) Nat Rev. Mol. Cell. Biol. 3: 673-684).

Transcriptional coactivator CBP (CREB binding protein) interacts withand regulates the activities of a multitude of signal-responsivetranscription factors and may thus integrate converging gene-regulatorypathways (Goodman and Smolik (2000) Genes Dev. 14: 1553-1577). CBP actsas a scaffold that facilitates recruitment of additional transcriptionalmodulators on the basal transcriptional complex. In addition, CBP has anintrinsic histone acetyltransferase (HAT) activity that may be used toregulate transcription by acetylating chromatin (Bannister andKouzarides (1996) Nature 384: 641-643). CBP regulates many physiologicalprocesses including cell growth, differentiation, and apoptosis. Changesin CBP activities are associated with a large number of developmental,neurodegenerative and mental retardation conditions, including the humanRubenstein-Taybi syndrome, and overexpression of Drosophila CPB can leadto neurodegeneration suggesting that cellular levels of CBP are tightlyregulated. CBP is a coactivator of transcription factor CREB (cyclic AMPresponse element binding protein) that regulates the expression of avariety of genes that contain CREs (cyclic AMP response elements) intheir promoters. CREB is implicated in a number of cellular processesand diseases (Mayr and Montminy (2001) Nat. Rev. Mol. Cell. Biol. 2:599-609) and CREB-dependent gene expression is critical for the functionand plasticity of the nervous system (Lonze and Ginty (2002) Neuron 35:605-523) including long-term memory and learning in both vertebrates andinvertebrates (Kandel (2001) Science 294: 1030-1038). Stimulation ofCREB-mediated transcription requires phosphorylation at CREB-Ser133, anevent that leads to the recruitment of CBP and stimulation oftranscription (Chrivia et al. (1993) Nature 365: 855-859).

In accordance with the present invention it has been discovered thatapoptosis or Ca⁺⁺ influx stimulates a PS1/γ-secretase-like cleavage ofE-cadherin. It has been further discovered that PS1 binding toE-cadherin is required for PS1/γ-secretase-like cleavage of E-cadherin.This cleavage results in the release of the cytoplasmic sequence ofE-cadherin, β-catenin and α-catenin to the soluble cytosol, thusfacilitating disassembly of cadherin-based adherens junctions. It hasfurther been discovered that peptides based upon the PS1 binding site ofcadherin inhibit γ-secretase activity, and are thus useful forinhibiting amyloid formation. In addition, it has been discovered thatPS1 promotes a γ-secretase-like, or ε-cleavage of N-cadherin. Thiscleavage results in the production of a soluble intracellular domain(ICD) fragment termed N-Cad/CTF2. This peptide fragment binds CBP andsequesters it to the cytoplasm thus decreasing nuclear CBP andsuppressing CREB-mediated transcription.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to peptides and polypeptides thatcomprise the presenilin-1 (PS1) binding site of cadherin and functionalequivalents thereof. In a preferred embodiment, the polypeptidecomprises the cytoplasmic domain of cadherin or a fragment thereofcapable of binding to PS1. In another preferred embodiment, the peptideor polypeptide comprises the sequence EGGGE (SEQ ID NO: 5). Compositionscomprising the peptides or polypeptides are also provided.

In another embodiment, the invention is directed to a method ofinhibiting PS1-mediated γ-secretase activity comprising contacting acell capable of exhibiting such activity with a peptide or polypeptidecomprising the PS1-binding site of cadherin or a functional equivalentthereof.

In another embodiment, the present invention provides a method ofpreventing or inhibiting amyloid deposition comprising administering toa subject in need of such treatment a composition comprising a peptideor polypeptide comprising the PS1-binding site of cadherin or afunctional equivalent thereof.

The present invention is also directed to peptides and polypeptides thatcomprise the matrix metalloproteinase (MMP) cleavage site of cadherinand functional equivalents thereof, and compositions comprising suchpeptides or polypeptides. A method of inhibiting metastasis comprisingadministering a composition comprising such a peptide or polypeptide isalso provided.

In another embodiment, the present invention provides a method ofdetermining susceptibility to Alzheimer's disease. A method ofidentifying agents that modify PS1/γ-secretase-like processing ofcadherin is also provided.

In another embodiment, the present invention provides a method oftreating FAD comprising administering to a subject in need of suchtreatment a composition comprising an agent that increases levels ofCad/CTF2. Compositions comprising Cad/CTF2 are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram depicting a portion of the amino acid sequence ofhuman E-cadherin (SWISS-PROT ACCESSION NO. P12830) (SEQ ID NO:19)indicating the N-termini of E-Cad/CTF1 and E-Cad/CTF2. Arrows identifythe cleavage sites of MMP and PS1/γ-secretase-like cleavage. Thesequence mediating E-cadherin-PS1 binding identified by Baki et al.(2001) Proc. Natl. Acad. Sci. USA 98:2381-2386 is underlined. EC1-5denote the extracellular E-cadherin repeats. TM denotes thetransmembrane domain.

FIG. 2 is a dose-response graph demonstrating that the production ofamyloid-β (1-40) decreases with increasing expression of E-cadherin.

FIG. 3 is a bar graph depicting the production of amyloid-β (1-42) incultures of control (L) and E-cadherin-transfected (EL) cells.

FIG. 4A is a bar graph depicting the production of amyloid-β (1-40) incultures of control (L) and E-cadherin-transfected (EL) cells. FIG. 4Bis a bar graph demonstrating that cytoplasmic E-cadherin (E-Cad/CTF-2)inhibits Aβ₄₀ and Aβ₄₂ in CHO cells expressing Swedish APP mutant

FIG. 5 is a Western blot depicting cadherin and cadherin cleavageproducts in cells transfected with wild-type and mutant PS1.

FIG. 6 is a Western blot depicting N-cadherin and N-cadherin cleavageproducts in fibroblasts from PS1 wild-type and PS1 P264L homozygousknock-in mice.

FIGS. 7A-D are Western blots depicting N-cadherin, N-cadherin cleavageproducts and PS 1 in extracts from PS1+/+ and PS1−/− embryos (FIG. 7A),PS1+/+ and PS1−/− fibroblasts (FIG. 7B), membranes from PS1+/+ andPS1−/− fibroblasts and N2a cell cultures (FIG. 7C) and membranes fromHEK293 cells transfected with wild type PS1 and D257A-PS1.

FIGS. 8A and B are Western blots depicting N-cadherin, N-cadherincleavage products and PS1 in membranes of PS1+/+, PS1−/− and PS1+/−mouse brain neuronal cultures (FIG. 5A) and membranes of rat brainneurons preincubated in the absence or presence of D-APV and stimulatedwith KCl, L-glutamate or NMDA.

FIGS. 9A and B are bar graphs depicting the relative CRE-dependentluciferase activity in L cells transfected with increasing amounts ofpN-Cad/CTF2 (FIG. 9A) and HEK293 cells transfected with PS1 (FIG. 9B).FIG. 9C depicts relative levels of c-fos or gapdh mRNAs in L cellstransfected with pN-Cad/CTF2. FIG. 9D is a Western blot depicting c-fosand N-Cad/CTF2 in L cells transfected with pN-Cad/CTF2.

FIG. 10A is a graph depicting CRE-dependent transactivation in N2a cellsincubated with DMSO or L-685,458. FIG. 10B depicts c-fos and gadph mRNAin N2a cells treated with DMSO or L-685,458. FIGS. 10C-E are Westernblots depicting c-fos in extracts of N2a cells treated with DMSO orL-685,458 (FIG. 10C), extracts of PS1+/+ or PS1−/− mouse fibroblasts(FIG. 10D), and extracts of PS1+/− and PS1−/− mouse embryonic brains(FIG. 10E).

FIGS. 11A and B are Western blots of L cells transfected withpN-Cad/CTF2. FIG. 11C depicts the results of an electrophoretic mobilityshift assay that examined the effect of N-Cad/CTF2 on the formation of aCBP/CREB/DNA complex.

FIGS. 12A and B are Western blots of membranes of HEK293 cellstransfected with WT-PS1 and PS1 mutants. FIG. 12C shows densiometricquantitation of N-Cad/CTF2 in each mutant. FIG. 12D depictsCRE-dependent transactivation for each mutant.

FIGS. 13A and B are Western blots of membranes (FIG. 13A) and proteinextracts (FIG. 13B) from embryonic fibroblast culture of wild type andPS1 P264L knock-in mice. FIG. 13C is a schematic representation of theeffects of PS1/ε-cleavage of N-cadherin on CBP/CREB signaling.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, it has been discovered thatbinding of PS 1 to the cytoplasmic domain of E-cadherin is required forprocessing of E-cadherin by the PS1/γ-secretase system. Further, it hasbeen discovered that N-cadherin and VE-cadherin are also metabolized bythe PS1/γ-secretase system. PS1 is involved in the processing of othercell surface transmembrane proteins including APP. By inhibiting thebinding of PS1 to its substrate, proteolytic processing of the substrateis prevented. The present invention provides polypeptides and peptidesthat inhibit the binding of PS1 to its substrate, and thereby preventproteolytic processing. Since the processing of APP is correlated withamyloid formation, the present peptides and polypeptides are useful ininhibiting amyloid formation and in the treatment and prevention ofdisorders characterized by amyloid formation.

In one embodiment, the present invention provides peptides andpolypeptides that comprise the PS1 binding site of cadherin. The termcadherin, as used herein, includes epithelial (E-), neural (N-),vascular endothelial (VE-), and other homologous cadherins. The cadherinis preferably a mammalian cadherin, and more preferably a humancadherin. Cadherins and the amino acid sequences thereof are known inthe art. As used herein, references to cadherin sequences are based uponthe numbering of the unprocessed cadherin precursor. Amino acidsequences of cadherins are available in publicly accessible databases.For example, the amino acid sequence of human VE-cadherin is availableat SWISS-PROT accession No. P33151. The amino acid sequence of humanN-cadherin is available at SWISS-PROT accession No. P19022. The aminoacid sequence of E-cadherin is available at SWISS-PROT accession No.P12830.

A polypeptide comprising the cytoplasmic domain of cadherin may be, forexample, a polypeptide comprising amino acids 621-784 of VE-cadherin(P33151), a polypeptide comprising amino acids 647-906 of N-cadherin(P19022), or a polypeptide comprising amino acids 732-882 of E-cadherin(P12830). Those of ordinary skill in the art can identify polypeptidescomprising the cytoplasmic domains of other cadherins. Fragments ofthese polypeptides that are capable of binding PS1 are also encompassedby the present invention. For example, a domain comprising amino acids758-769 of E-cadherin is capable of binding PS1 as disclosed by Baki etal. (2001) Proc. Natl. Acad. Sci. USA 98:2381-2386, the disclosure ofwhich is incorporated herein by reference. A preferred peptide of thepresent invention has the sequence EGGGEEDQDFDL (SEQ ID NO.: 1). Ahomologous peptide derived from the sequence of VE-cadherin has thesequence EGGGEMDTTSYD (SEQ. ID NO.: 2). A homologous peptide derivedfrom the sequence of N-cadherin has the sequence EGGGEEDQDYDLS (SEQ. IDNO. 3). Those of ordinary skill in the art can determine other peptidescapable of binding to PS1 by examining sequence similarities in othercadherins, or by conducting PS1 binding assays, or by performingdeletion analysis as taught by Baki et al. In a preferred embodiment,the polypeptide or peptide comprises the sequence EGGGEED (SEQ. ID. NO.:4). In another preferred embodiment, the polypeptide or peptidecomprises the sequence EGGGE (SEQ. ID. NO.: 5)

Functional equivalents of the foregoing polypeptides and peptides arealso provided by the present invention. Functional equivalents aredefined herein as variants that maintain the ability to bind to PS1,including substitutions, insertions, deletions, additional sequencessuch as targeting sequences, tags, labeled residues, sequences toincrease half-life or stability, or residues for any other purpose solong as the peptide maintains the ability to bind to PS1. The aminoacids may be naturally occurring or modified, and may be L-amino acidsor D-amino acids. The polypeptides and peptides of the invention may bemodified, for example, by acylation or amidation. Peptide backbones maybe modified, for example by substituted amide linkages.

The term functional equivalents also includes mimetics of the peptidesand polypeptides of the present invention. Peptide mimetics are known tothose of ordinary skill in the art and include peptides or non-peptidesmall molecules that have the activity of the peptide or polypeptide onwhich they are modeled. The design of such mimetics is based uponstructure function studies of the peptides and polypeptides of theinvention. Methods of determining protein structure are known in the artand include approximation by analogy to related proteins and othertechniques including X-ray crystallography and computer modelingstudies. These studies are used to design molecules that mimic the shapeand function of the template peptide or polypeptides, which can then besynthesized by methods known in the art.

The peptides of the present invention are preferably from about six toabout fifteen amino acids in length, and more preferably from about tento about thirteen amino acids in length. Peptides may be modified toincrease cell permeability, for example by linking the peptides to cellpermeant peptide vectors such as Antennapedia (43-58), Arg/Trp analogue,TAT (48-60) and kFGF hydrophobic signal peptide region as described byDunican et al. (2001) Biopolymers 60:45-60.

The peptides and polypeptides of the present invention may besynthesized by methods known in the art. Peptides may be synthesized,for example, by solid-phase methodology on an automated peptidesynthesizer. Peptides may also be prepared by use of a combinatorialpeptide library by methods known in the art. Polypeptides and peptidesmay also be prepared by recombinant methods, for example, by preparingexpression vectors containing DNA encoding the desired polypeptide orpeptide, transforming host cells with the vectors, culturing host cellsunder conditions whereby the polypeptide or peptide is expressed, andrecovering the recombinant product.

The present invention further provides compositions comprising thePS1-binding peptides and polypeptides or functional equivalents thereof.The peptide, polypeptide or functional equivalent thereof may be in theform of a pharmaceutically acceptable salt. The compositions compriseone or more peptides or polypeptides or functional equivalents includingmimetics as described above and may further comprise a carrier ordiluent including for example solvents, dispersion media, antibacterialand antifungal agents, microcapsules, liposomes, cationic lipidcarriers, isotonic and absorption delaying agents and the like.

In another embodiment, the present invention provides a method ofinhibiting PS1-mediated γ-secretase activity comprising contacting acell capable of exhibiting such activity with a composition comprising apeptide or polypeptide comprising the PS1-binding site of cadherin or afunctional equivalent thereof.

PS1-mediated γ-secretase processing of APP is correlated with amyloidformation, and thus inhibition of PS1-mediated γ-secretase activity isuseful for preventing or inhibiting amyloidosis, and thereby treating orpreventing Alzheimer's disease. The invention provides a method ofpreventing or inhibiting amyloid deposition comprising administering toa subject in need of such treatment a composition comprising a peptideor polypeptide comprising the PS1-binding site of cadherin or functionalequivalents thereof, in an amount effective to prevent or inhibitamyloid deposition.

The present invention further provides peptides and polypeptides thatcomprise the MMP cleavage site of cadherin and functional equivalentsthereof. In accordance with the present invention, it has beendiscovered that the MMP cleavage site of human E-cadherin is at residues700-101, i.e., seven residues to the extracellular side of thetransmembrane domain of E-cadherin. This cleavage site is closer to theextracytoplasmic face of the plasma membrane than previously reported byIto et al. (1999) Oncogene 18:7080-7090.

Peptides that comprise the MMP binding site of cadherin include peptidesderived from the extracellular domain of cadherins adjacent to thetransmembrane domain. Such peptides may comprise the sequencesCEGAAQVCRKAQPVEAGLQI (SEQ. ID. NO.: 6) derived from E-cadherin;CDSNGDCTDVDRIVGAGLGTG (SEQ. ID. NO.: 7) derived from N-cadherin; andKCNEQGEFTFCEDMAAQVGVS (SEQ. ID. No.: 8) derived from VE-cadherin, andthe corresponding regions of other cadherins. In a preferred embodiment,the peptide comprises the sequence KAQPVEAGLQI (SEQ. ID. NO.: 9). Inanother preferred embodiment the peptide comprises the sequence QPVEA(SEQ. ID. NO.: 10). Fragments of these peptides and functionalequivalents are also included. Functional equivalents are defined hereinas variants that are capable of inhibiting cleavage of cadherin by MMP,and include the variants and modifications described hereinabove for thePS1-binding peptides and polypeptides, as well as mimetics as describedhereinabove. The ability of the peptides and polypeptides to inhibitcleavage of cadherin can be assessed by contacting cultured cells withthe peptide or polypeptide and assaying for cadherin cleavage, forexample by immunoassays using antibodies against the MMP cleavageproducts of cadherin. The peptides may be synthesized as describedhereinabove. The peptides are preferably from about six to fifteen, andmore preferably from about ten to thirteen amino acids in length.

The present invention provides compositions that comprise peptides thatcomprise the MMP cleavage site of cadherin and functional equivalentsthereof, such as mimetics. The compositions may further comprise acarrier or diluent as described hereinabove.

In tumor metastasis, inhibition of cadherin-mediated cell adhesion ispromoted by cleavage of cadherin by MMP. The present polypeptides andpeptides block cleavage of cadherin by MMP, and thus are useful ininhibiting metastasis. Accordingly, in another embodiment, the presentinvention provides a method of inhibiting tumor metastasis comprisingadministering to a subject in need of such treatment a compositioncomprising a polypeptide or peptide comprising the MMP cleavage site ofcadherin or a functional equivalent thereof, in an amount effective toinhibit tumor metastasis. Inhibition of tumor metastasis can be assessedby those of ordinary skill in the art, for example, by conventionalimaging methods.

Further, processing of cadherin by MMP results in CAJ disassembly,leading to apoptosis. The present polypeptides and peptides block thisprocessing and thereby inhibit apoptosis. Accordingly, the presentinvention further provides a method of inhibiting apoptosis comprisingcontacting cells undergoing apoptosis with a composition comprising apolypeptide or peptide comprising the MMP cleavage site of cadherin or afunctional equivalent thereof in an amount effective to inhibitapoptosis.

The polypeptides or peptides or their functional equivalents may beadministered in a composition further comprising a pharmaceuticallyacceptable carrier. The formation of pharmaceutical compositions isknown in the art and disclosed for example in Remington's PharmaceuticalSciences, 17th edition, Mack Publishing Co., Easton, Pa. Polypeptide andpeptide modifications as described above, for example peptides linked tocell permeant vectors, are particularly preferred for pharmaceuticalcompositions. The polypeptides, peptides or their functional equivalentsmay also be delivered by methods of gene therapy known in the art.Nucleic acids encoding the peptide or polypeptide are inserted intovectors such as genetically engineered adenovirus, adenoassociatedvirus, or herpes virus vectors, and the vectors are delivered to thesubject in the form of a pharmaceutical composition. Nucleic acidsencoding the peptides, polypeptides or functional equivalents may alsobe delivered by non-viral gene transfer systems such as liposome-DNAcomplexes or receptor-mediated gene transfer.

The effective amount of the peptide, polypeptide or functionalequivalent to be used in the methods of the invention can be determinedby the skilled artisan with consideration of individual differences inage, weight, extent of disease and condition of patient. Thepharmaceutical forms containing the active agents may be administered inany convenient manner, either orally or parenterally, such as byintravenous, intraperitoneal, subcutaneous, rectal, implant,transdermal, slow release, intrabuccal, intracerebral or intranasaladministration. Generally, the active agents need to pass the bloodbrain barrier and may have to be chemically modified, e.g. madehydrophobic, or linked to cell permeant vectors to facilitate this or beadministered directly to the brain or via other suitable routes. Forinjectable use, sterile aqueous solutions are generally used oralternatively sterile powders for the extemporaneous preparation ofsterile injectable solutions may be used. The solutions must be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms such as bacteria andfungi. The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propylene glycoland liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thirmerosal and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activeagents in the required amount in the appropriate solvent with various ofthe other ingredients enumerated above, as required, followed bysterilization by, for example, filtration or irradiation. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and the freeze-dryingtechnique which yield a powder of the active ingredient plus anyadditional desired ingredient from previously sterile-filtered solutionthereof.

When the active agents are suitably protected they may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or may be enclosed in hard or soft shell gelatincapsule, or may be compressed into tablets, or may be incorporateddirectly with the food of the diet. For oral therapeutic administration,the active compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like.

As disclosed hereinabove, the PS1/γ-secretase system promotes classicγ-secretase cleavages of APP defined by the C-terminus of various Aβspecies. In addition, it promotes a γ-secretase-like cleavage of severaltransmembrane proteins, including cadherin. The γ-secretase-likecleavage takes place further downstream from the amyloidogenicγ-secretase cleavages, at a site closer to the membrane/cytoplasminterfaces than the γ-cleavages. The γ-secretase-like cleavage is alsodesignated ε-cleavage, and those terms are used interchangeably herein.

The γ-secretase-like cleavage, or ε-cleavage, results in the productionof an intracellular domain (ICD) cleavage product. The ICD cleavageproduct is also referred to herein as Cad/CTF2 and the ε-cleavageproduct.

Certain mutations of PS1 are known to result in the development of aspecific form of Alzheimer's disease known as Familial Alzheimer'sdisease (FAD). It has been reported that these mutations increase theproduction of amyloid-β. Hardy (1997) Trends Nerosci. 20:154-159.However, it has not heretofore been clear how these mutations cause FAD.In accordance with the present invention it has been discovered that PS1mutants are impaired in their ability to mediate γ-secretase-like, orε-cleavage of N-cadherin and other cadherins. This impairment results inreduced production of the N-cadherin ICD cleavage product (N-Cad/CTF2).The production of the C-terminal MMP cleavage product N-Cad/CTF1 is notimpaired.

It has further been discovered in accordance with the present inventionthat the 6-cleavage product of N-cadherin (e.g. N-Cad/CTF2) bindstranscription factor CBP (CREB binding protein) and sequesters CBP tothe cytoplasm. Sequestration of CBP to the cytoplasm results ininhibition of CRE-dependent transactivation and inhibition of expressionof CBP/CREB-regulated genes such as c-fos. It has also been discoveredin accordance with the present invention that PS1 FAD mutationsincluding M146L, A246E, E280A, ΔE9, G384A, E280G and Y115H inhibitN-Cad/CTF2 production and upregulate CREB-mediated transcription. TheFAD mutation thus cause a gain of transcriptional function by inhibitingproduction of the transcriptional repressor N-Cad/CTF2.

Accordingly, the present invention provides a method of determiningsusceptibility to Alzheimer's disease comprising measuring the ICDproduct of the c-cleavage of cadherin in a cell, wherein a reduction inthe cleavage product relative to the levels in control cells from normalindividuals is indicative of susceptibility to Alzheimer's disease.

In a preferred embodiment, the ICD ε-cleavage product that is measuredis N-Cad/CTF2 or E-Cad/CTF2 or VE-Cad/CTF2. E-Cad/CTF2, depicted in FIG.1 (SEQ ID NO:19), comprises amino acids 732-882 of human E-cadherin(SWISS-PROT P12830). N-Cad/CTF2 comprises amino acids 747-906 of humanN-cadherin (SWISS-PROT P19022). VE-Cad/CTF2 comprises amino acids621-784 of VE-cadherin (SWISS-PROT P33151). The ICD cleavage products ofother cadherins are defined as the C-terminal portion of cadheringenerated by the γ-secretase-like ε-cleavage. The production of the ICDε-cleavage product may be assessed in cells obtained from tissue of apatient, including non-neural tissue such as skin, or from cell culturesestablished from a patient.

Reduced levels of Cad/CTF2 may indicate that the patient has an FAD PS1mutation and is therefore at risk of developing FAD. Alternatively,there may be reduced Cad/CTF2 in the absence of a FAD PS1 mutationindicating a risk of developing sporadic AD. Cad/CTF2 may be measured byconventional methods of protein detection. A detectable reduction inCad/CTF2 relative to controls by such methods is sufficient for thepresent method. The measurement need not be quantitative. In a preferredembodiment, one of the many immunoassays well-known in the art is usedto detect and qualitatively measure Cad/CTF2. Suitable assays includeELISA, Western blotting, and radioimmunoassay. For example, cellextracts may be probed by conventional Western blots using ananti-cytoplasmic N-cadherin or anti-cytoplasmic E-cadherin antibody oranti-cytoplasmic VE-cadherin antibody. Such an antibody will recognizeboth the Cad/CTF1 and Cad/CTF2 fragments, which are distinguishable onWestern blots by their relative molecular weights, with the Cad/CTF2fragment having a lesser molecular weight. Such antibodies may beobtained commercially (e.g. C36 from BD Transductor Laboratories) orraised by conventional methods known in the art, disclosed for examplein Antibodies: A Laboratory Manual, Harlow et al., eds, Cold SpringHarbor Laboratories, 1988.

The present invention further provides a method for identifying agentsthat modify PS1/γ-secretase-like processing of cadherin. Such agents areuseful as candidate compounds for treatment of conditions implicated incadherin processing, including for example Alzheimer's disease. Themethod comprises contacting a cell containing cadherin with a testcompound; measuring production of the ICD cleavage product of cadherin;and comparing production of the cleavage product in cells contacted withthe test compound to production in cells not contacted with the testcompound; wherein a difference in production of the cleavage product inthe presence of the test compound is indicative of an agent thatmodifies γ-secretase-like processing of cadherin. In accordance with thepresent invention it has been found that the ionophore ionomycinmodifies PS1/γ-secretase-like processing of cadherin and is thus acandidate compound for treatment of conditions implicated in cadherinprocessing.

The assay may be in vitro or in vivo. For example, the cell may be acultured cell or a cell obtained from a mammal to which the testcompound has been administered. Suitable cell lines include mammalian,preferably human cells such as HEK293, HeLa cells, primary humanenthothelial cells, primary human fibroblasts or lymphoblasts, primaryhuman mixed brain cells, Chinese hamster ovary cells, and the like.

The mammal may be, for example, a monkey, dog, rabbit, guinea pig, rator mouse. In a preferred embodiment the mammal may be an animal modelfor Alzheimer's disease, such as a PS1 P264L homozygous knock-in mousedescribed by Siman et al. (2000) J. Neurosci. 20:8717-26. The compoundmay be administered in a composition and by a route of administration asdescribed hereinabove.

In a preferred embodiment the cell is from a cultured human cell line.The cells may be cells that overproduce cadherin, for example as aresult of transfection with cadherin cDNA. The cell may be a cell inwhich PS1 contains one or more mutations for example a cell transfectedwith a mutant PS1 known to be correlated with FAD.

Measurements of the γ-secretase-like cleavage product of cadherin, i.e.the soluble ICD fragment termed Cad/CTF2, may be accomplished by anytechnique capable of detecting Cad/CTF2. Immunological detection methodsusing binding substances for Cad/CTF2 such as antibodies and antibodyfragments are preferred. Suitable detection methods include ELISA,Western blotting and radioimmunoassay. As discussed hereinabove,antibodies that recognize Cad/CTF2 cross-react with Cad/CTF1, and thusWestern blotting or other methods that permit separation of Cad/CTF2 andCad/CTF1 are preferred.

The test compound may be any agent that can be added to the cell withoutsubstantially interfering with cell viability, including for examplesmall molecules, polymers including polypeptides, polysaccharides,polynucleotides and the like, and may be natural or synthetic, and maybe a single substance or a mixture, for example a cell extract.Ionophores, and in particular calcium ionophores such as ionomycin andA23187, are specifically contemplated. Also contemplated are agonists ofionotropic receptors, such as the N-methyl-D-aspartate (NMDA) receptor.Such agonists include NMDA, 1-aminocyclobutane-1,3-dicarboxylic acid,aspartic acid, 2-carboxy-3-carboxy methylquinoline, cysteinesulphinicacid, glutamic acid, homoquinolinic acid,“-amino-(3-hydroxy-5-isoxazolyl)acetic acid and tetrazol-5-yl-glycine.

In another embodiment, the present invention provides a method ofidentifying agents that modify CREB-mediated transcription. Such agentsare useful as candidate compounds for treatment of conditions implicatedin unregulated CREB-mediated transcription, such as FAD. The methodcomprises contacting a cell capable of undergoing CREB-mediatedtranscription with a test compound; measuring CREB-mediatedtranscription, and comparing CREB-mediated transcription in cellscontacted with the test compound to CREB-mediated transcription in cellsnot contacted with the test compound; wherein a difference inCREB-mediated transcription in the presence of the test compound isindicative of an agent that modifies CREB-mediated transcription.

The assay may be performed in vitro or in vivo, using cells, mammals andtest compounds as described above. A detectable difference inCREB-mediated transcription relative to controls is sufficient for thepresent method.

CREB-mediated transcription may be measured by using cells transfectedwith a reporter gene under the control of a promoter containing a CRE,and measuring expression of the reporter gene. CREB-mediatedtranscription may also be assessed by measuring expression of genesknown to be regulated by CREB, e.g. c-fos. A change in the amount ofc-fos mRNA or c-fos protein in the absence of an effect on transcriptionof genes that are not regulated by CREB, e.g. gadph or β-tubulin, isindicative of the identification of an agent that modifies CREB-mediatedtranscription. The mRNA and protein levels may be measured by methodsknown in the art, such as semiquantitative RT-PCR and Western blotting.Test compounds that modify CREB-mediated transcription includeionophores, NMDA receptor agonists, and Cad/CTF2.

The present invention further provides agents that modify CREB-mediatedtranscription. In a preferred embodiment, the agent binds to CBP andsequesters CBP to the cytoplasm. In a preferred embodiment the agent isthe ICD cleavage product of cadherin. In another preferred embodimentthe agent is N-Cad/CTF2, E-Cad/CTF2 or VE-Cad/CTF2. Such agents can beproduced as described hereinabove, e.g. by standard methods ofrecombinant technology, or obtained as cleavage products isolated fromthe naturally occurring mammalian proteins. Compositions comprising theagents are also provided, and may be formulated as describedhereinabove.

The present invention further provides a method for treating FADcharacterized by decreased Cad/CTF2 production. The method comprisesadministering to a subject in need of such treatment a compositioncomprising an agent that increases levels of Cad/CTF2 in the subject. Inanother embodiment, the method comprises administering an agent thatdecreases CREB-mediated transcription in a subject. In a preferredembodiment, the subject exhibits a PS1 FAD mutation associated withdecreased N-Cad/CTF2 production and upregulated CREB-mediatedtranscription as characterized hereinabove. The agent may be, e.g.,Cad/CTF2 itself or functional equivalents thereof that maintain theability to bind CBP and sequester it to the cytoplasm. Functionalequivalents are defined hereinabove. In a preferred embodiment the agentis N-Cad/CTF2. The agent may also be PS1, or functional equivalents asdefined above that maintain the ability to cleave cadherin and produceCad/CTF2. The agent may also be an agent that stimulates PS1 ε-cleavageactivity, such as an NMDA receptor agonist or other agent defined by theassay described above. The agents may be modified to increase cellpermeability as described above. Compositions may be formulated andadministered as described hereinabove.

All references cited herein are incorporated herein in their entirety.

The following examples serve to further illustrate the presentinvention.

Example 1 Materials and Methods

The following materials and methods were used in examples 2-7.

Materials and antibodies. GM6001 was obtained from ChemiconInternational, Inc., Staurosporine was from Sigma, lonomycin andZ-DEVD-FMK were from Calbiochem. L-685,458 and its inactive analoguewere kindly provided by Dr. M. S. Shearman (Merck Research Labs). Rabbitpolylonal antibody R222 was raised against amino acids 2-12 of humanPS1/NTF and mouse monoclonal antibody 33B10 is specific for residues331-350 of human PS1/CTF (Georgakopoulos et al., (1999) Mol. Cell.4:893-902. Rabbit polyclonal antibody R1 was raised against human APP751amino acids 729-751 (Anderson et al., (1989) Embo. J. 8:3627-3632).Anti-E-cadherin (clone C36), anti-β-catenin and anti-α-cateninmonoclonal antibodies were obtained from BD Transduction Laboratories;antibody H108 against E-cadherin ectodomain was obtained from Santa CruzBiotechnology, Inc.

Mouse embryo preparation. Wild-type and PS1 knock-out mouse embryos(Baki et al., (2001) Proc Natl. Acad. Sci. USA 98:2381-2386) werecollected at day 18.5 post coitum and solubilized by mechanicaldissociation and sonication in RIPA buffer (50 mM Tris-HCl [pH 8], 150mM NaCl, 0.1% SDS, 1% Nonidet P40, 0.5% sodium deoxycholate, 1× Completeprotease inhibitor cocktail, Roche). Fifty micrograms of extract wasanalyzed by Western blotting.

Cell Cultures and transfections. Cells were grown in Dulbecco's ModifiedEagle Medium (DMEM) plus 10% fetal bovine serum, penicillin andstreptomycin in 5% CO₂ at 37° C. Fibroblast cell lines derived eitherfrom wild-type (WT) (PS1+/+) or PS1 knock-out (PS1−/−) mice were stablytransfected with human E-cadherin as described by Baki et al., (2001).A431 cells were from American Type Culture Collection. Stabletransfectants of PS1 cDNA in HEK293 cells were prepared as described byGeorgakopoulos et al., (1999). A431D cells stably transfected withwild-type or GGG759-761AAA mutant E-cadherin were provided by Dr. A. B.Reynolds.

Subcellular fractionation. Confluent A431 cells (one 100 mm dish) wererinsed and scraped with 4° C. phosphate-buffered saline (PBS). Cellswere then placed into 1 ml of buffer A (20 mM Tris-HCl [pH 7.5], 0.25 Msucrose, 10 mM EGTA, 2 mM EDTA, 1× Complete protease inhibitor cocktail,Roche), passed through a 27 gauge needle 10 times, and the obtained celllysate was centrifuged at 500×g for 10 min at 4° C. The supernatant wasthen centrifuged at 120,000×g for 45 min at 4° C. to separate thecytosolic and crude membrane fractions. The pellet (crude membranefraction) was washed twice with buffer A and resuspended by sonicationin 400 ml of buffer A containing 1% Triton X-100. The suspension wasincubated at 4° C. for 30 min and then centrifuged at 120,000×g for 45min at 4° C. to separate the membrane and Triton X-100-insolublefractions. The pellet (Triton X-100-insoluble fraction) was washed twicewith buffer A and solubilized by sonication in RIPA buffer. 20 mg ofproteins from the different fraction were analyzed by SDS-PAGE.

Immunoprecipitations (IPs), immunoblotting, immunofluorescence andconfocal microscopy. For WB analysis of cell extracts, cells were washedwith PBS and then solubilized in RIPA buffer. For IPs cells weresolubilized in Hepes buffer (25 mM Hepes [pH 7.4], 150 mM NaCl, 1×Complete protease inhibitor cocktail, Roche) containing 1% digitonin(IPs with I-R222 and PI-R222 antibodies) or 1% Triton X-100 (IPs withβ-catenin and desmoglein antibodies). Following centrifugation at17,000×g for 10 min, supernatants (1 mg protein) were pre-cleared withprotein A- or protein G-agarose (Pierce) for 2 hours. Supernatants werethen incubated with antibodies overnight at 4° C. and treated for 2hours with protein A-agarose (polyclonal antibodies) or with proteinG-agarose (monoclonal antibodies). IPs were washed with Hepes buffercontaining either 1% digitonin or 1% Triton X-100 and analyzed bySDS-PAGE. Immunofluorescence and confocal microscopy was performed asdescribed by Georgakopoulos et al., (1999). Briefly, cells were platedon 22×22 mm collagen-coated glass coverslips, fixed in cold methanol for10 min at −20° C. Following washing in TBS (25 mM Tris-HCl [pH 7.4], 150mM NaCl), cells were treated with 10% goat serum in SuperBlock (Pierce)for 1 hour, incubated overnight with primary antibody (1:100), washed inTBS and then were incubated with species specific Alexa Fluor™ secondaryantibody conjugates (Molecular Probes). Cells were washed with TBS,mounted with Prolong antifade kit (Molecular Probes), and photographedon a Leica confocal laser scanning microscope.

Purification, mass spectrometry, and amino-terminal sequence analyses ofE-cadherin carboxyl-terminal fragments. A431 cells treated with 1 mM ofstaurosporine for 6 hours were solubilized by sonication in RIPA buffer.50 mg of protein extract were pre-cleared with 20 mg of H108 antibody,20 mg of unrelated monoclonal antibody and a mixture of protein A- andprotein G-agarose (Pierce). The pre-cleared supernatant was treatedovernight with 120 mg of C36 anti-E-cadherin antibody andimmunoglobulins were precipitated with 500 ml of protein G-agarose.Immunoprecipitates were split in two samples and submitted to SDS-PAGE.One sample was stained with GelCode blue stain reagent (Pierce) and the33 kDa and 38 kDa fragments of E-cadherin were submitted to MALDI-MSPeptide Mass Mapping after in-gel digestion (Dr. M. A. Gawinowicz,HHMI/Columbia University Protein Core Facility). The second sample wastransferred on to a polyvinylidene difluoride (PVDF) membrane and the 38kDa and 33 kDa bands corresponding to E-Cad/CTF1 and E-Cad/CTF2respectively, were subjected to sequential derivitization and cleavageof N-terminal amino acids by Edman chemistry followed by reverse phaseHPLC chromatography (automated Applied Biosystems Procise 492 PeptideSequencer, New York University Protein Analysis Facility).

Example 2 A PS1/γ-Secretase-Like Activity Controls E-Cadherin Processing

Extracts from PS1-knock-out mouse embryos (PS1−/−) were used toinvestigate whether PS1 plays any role in E-cadherin processing.Extracts from PS1+/+ or PS1−/− mouse embryos were probed on Westernblots with either anti-cytoplasmic E-cadherin C36 or anti-cytoplasmicAPP R1 antibodies. PS1−/− embryos had significantly higher amounts of a38 kDa peptide that contained the cytoplasmic sequence of E-cadherin(E-Cad/CTF1) than did wild-type (WT, PS1+/+) embryos even though allembryos had similar levels of the full-length E-cadherin. PS1−/− embryosalso contained increased levels of APP α-stubs.

Extracts from E-cadherin-transfected PS1+/+ or PS1−/− mouse fibroblastswere probed with anti-E-cadherin C36 or 33B10 antibodies. E-Cad/CTF1accumulated in the fibrobast cell line that was derived from PS1−/− micecompared to the cell line from PS1+/+mice, even though both cell linesexpressed comparable amounts of transfected full-length E-cadherin. Theaccumulation of E-Cad/CTF 1 in PS1−/− cells under conditions of constantlevels of the full length protein indicated that a PS1-mediated activitycontrols metabolism of E-Cad/CTF1.

PS1+/+fibroblasts were treated for 6 hours either with the γ-secretaseinhibitor L-685,458 (0.5 μM) or with dimethylsulfoxide. Extracts fromthese cell cultures were then probed with anti-E-cadherin C36. Treatmentof the E-cadherin-transfected PS1+/+ fibroblasts with the selectiveγ-secretase inhibitor L-685,458 increased cellular E-Cad/CTF1 comparedto non-treated controls, indicating that the PS1-associated γ-secretaseactivity is involved in the metabolism of E-Cad/CTF1.

Although the foregoing data indicated that peptide E-Cad/CTF1 is furtherprocessed by a PS1/γ-secretase cleavage, E-cadherin metabolitesresulting from this activity either in embryos or in PS1+/+ fibroblastswere not detected. The apparent molecular weight and immunoreactivity ofE-Cad/CTF1, however, suggested that this fragment derives from a matrixmetalloproteinase (MMP) cleavage of the E-cadherin ectodomain. Sincethis cleavage is stimulated by apoptosis (Steinhusen et al., (2001) J.Biol. Chem. 276:4976-4980), the levels of the PS1/γ-secretase cleavageproduct of E-cadherin in apoptotic conditions was investigated. Humanepithelial cell line A431 that expresses high levels of endogenousE-cadherin and undergoes apoptotis under staurosporine treatment(STS)(Steinhusen et al., (2001)) was used as a model. A431 cells weretreated for 1, 2, 3, 4, 5 or 6 hours with 1 μM of STS to induceapoptosis, solubilized in RIPA and blotted with anti-E-cadherin C36antibody. STS treatment of this cell line resulted in a time-dependentproduction of three E-cadherin carboxy-terminal fragments migrating at38, 33, and 29 kDa respectively.

A431 cells were also preincubated for 30 minutes in the absence orpresence of GM6001 (2.5 μM), Z-DEVD-FMK (50 μM), an inactive analogue ofL-685,458 (0.5 μM), or L-685,458 (0.5 μM). Cells were then treated withSTS for 6 hours to induce apoptosis, and cell extracts were probed withC36 antibody. Production of the 38 kDa fragment was inhibited by the MMPinhibitor GM60001 (Galardy et al., (1994) Ann. N.Y. Acad. Sci.732:315-323) indicating that this fragment which has identicalimmunoreactivity and apparent molecular mass as E-Cad/CTF1 is derivedfrom a MMP cleavage of E-cadherin.

Conditional media (20 μl) from A431 cells cultured in the absence orpresence of GM60001 and treated with SDS as above were probed on Westernblots with anti-E-cadherin ectodomain antibody H108. STS increased asecreted 95 kDa fragment detected with the E-cadherin ectodomainantibody H108. This fragment (termed E-Cad/NTF1), does not react withantibodies against cytoplasmic E-cadherin and, like E-Cad/CTF1, it isalso inhibited by GM60001 suggesting that E-Cad/NTF1 is the secretedcounterpart of E-Cad/CTF1. The 29 kDa fragment (E-Cad/CTT3) is inhibitedby the specific caspase-3 inhibitor Z-DEVD-FMK indicating it is producedby an apoptosis-stimulated caspase-3 cleavage of E-cadherin (Steinhusenet al., (2001).

The γ-secretase inhibitor L-685,458 completely blocked production of the33 kDa cadherin fragment (E-Cad/CTF2) indicating that this fragment isproduced by a γ-secretase-like cleavage of E-cadherin. Inhibition ofE-Cad/CTF2 by L-685,458 correlates with an increase in E-Cad/CTF1indicating that the former peptide derives from the later by aγ-secretase-like activity.

Extracts of HEK293 cells stably transfected with PS1 or vector alonewere immunoprecipitated and probed with C36 antibody or anti-PS1/NTFantibody R222. Over-expression of PS1 in cell line HEK293 increasedE-Cad/CTF2 and decreased E-Cad/CTF1. That E-Cad/CTF2 is produced evenwhen E-Cad/CTF1 is inhibited indicates that thePS1/γ-secretase-dependent E-Cad/CTF2 can also be derived fromfull-length E-cadherin.

A431 cells were pre-incubated for 30 minutes in the absence or presenceof GM6001 (1.5 μM). Cells were then treated with STS for 6 hours andcell extracts were probed on Western blots with H108 or C36 antibodies.A 100 kDa E-cadherin fragment recognized by antibody H108 but not byanti-cytoplasmic E-cadherin antibody C36 was detected in cell extractsof GM60001-treated A431 cultures, indicating that this fragment(E-Cad/NTF2) is the N-terminal counterpart of E-Cad/CTF2. In addition toA431 and HEK293 cells, two other cell lines, SW480 and LNCaP, alsoproduced the PS1/γ-secretase fragment E-Cad/CTF2 under Ca++ influxconditions indicating that this is a general mechanism of E-cadherinprocessing.

Example 3 Identification of the PS1/γ-Secretase and MMP-MediatedCleavage Sites of E-Cadherin

E-Cad/CTF 1 and E-Cad/CTF2 were affinity purified from STS-treated A431cells. Antibody H108 against E-cadherin sequence 600-707 (numberingaccording to the full length unprocessed human E-cadherin) reacted withsecreted E-Cad/NTF1 but not with cellular E-Cad/CTF1 indicating that theMMP cleavage of E-cadherin occurs closer to the extracytoplasmic face ofthe plasma membrane than previously reported by Ito et al., (1999)Oncogene 18:7080-7090. Indeed, Edman sequencing of E-Cad/CTF1 through 14cycles showed the following major sequence: VEAGLQIPAILGIL (SEQ. ID NO.:11). This is a unique sequence corresponding to human E-cadherinresidues 701-714. The N-terminus of this sequence is located sevenresidues upstream of the transmembrane sequence of E-cadherin. Massspectrometry analysis of E-Cad/CTF1 showed no peptides upstream of thecleavage site that was determined by Edman sequencing. Thus, the 38 kDaE-Cad/CTF1 is produced by a MMP cleavage after E-cadherin residuePro⁷⁰⁰. Edman sequencing of E-Cad/CTF2 yielded the following sequence:RRRAVVKEPLL (SEQ. ID. NO.: 12). This is a unique sequence correspondingto human E-cadherin residues 732-742. Mass spectrometric analysis ofE-Cad/CTF2 yielded E-cadherin peptides predicted from the sequencingdata. These results show that the PS1-mediated γ-secretase cleavage ofE-cadherin takes place between residues Leu⁷³¹ and Arg⁷³² at theinterface of the membrane with the cytoplasm. The foregoing results aresummarized schematically in FIG. 1. The indicated γ-secretase-likecleavage is also called the E-cleavage that produces Cad/CTF2 fragments.

Example 4 The γ-Secretase-Mediated Cleavage of E-Cadherin PromotesDisassembly of Adherens Junctions

The molecular weight and immunoreactivity of the isolated peptidessuggest that they contain the entire E-cadherin cytoplasmic sequenceincluding the β-catenin binding site (Steinberg et al., (1999) Curr.Opin. Cell. Biol. 11:554-560). Extracts from STS-treated A431 cells wereimmunoprecipitated with antibodies against PS1 (I-R222), pre-immuneserum (PI-R222), β-catenin or desmoglein, and the immunoprecipitatesobtained were probed on Western blots with anti-E-cadherin antibody C36.These co-immunoprecipitation experiments showed that the MMP cleavageproduct E-Cad/CTF1 binds both β-catenin and PS1 whereas the γ-secretaseproduct E-Cad/CTF2 binds only β-catenin. Thus, the PS1/γ-secretase-likecleavage of E-cadherin dissociates PS1 from the E-cadherin/β-catenincomplex.

A431 cells treated for 6 hours with STS were fractionated into membrane,soluble cytosolic and Triton X-100-insoluble fractions, and thefractions were probed on Western blots with C36 antibody. Thissubcellular fractionation of STS-treated A431 cells showed thatfull-length E-cadherin and E-Cad/CTF1 are found only in the membrane andcytoskeletal (Triton X-100-insoluble) fractions while E-Cad/CTF2localizes in the membrane and in the soluble cytosol indicating that thePS1/-γ-secretase-like cleavage results in the solubilization of thecytoplasmic sequence of E-cadherin. In stable cell-cell adhesion, theE-cadherin/β-catenin complex of the CAJ is anchored to the actincytoskeleton via catenin and this association is manifested by theinsolubility of the complex components in Triton X-100 (Baki et al.,(2001)). Induction of apoptosis or calcium influx disruptscadherin-mediated cell-cell adhesion by cleaving cadherin anddisassembling the CAJ complexes. To determine whether the γ-secretasecleavage of E-cadherin is involved in the CAJ disassembly, calciuminflux was induced with ionomycin (10 μM) in A431 cells preincubated for30 minutes in the absence or presence of the γ-secretase inhibitorL-685,458. Cell extracts were fractionated, and analyzed on Westernblots with antibodies against cytoplasmic E-cadherin C36 or β- andα-catenins. In the absence of this inhibitor, ionomycin induced atime-dependent decrease in the cytoskeletal (Triton X-100-insoluble)fraction of both full-length E-cadherin and E-Cad/CTF1 and this decreasecorrelated with a corresponding increase in soluble cytosolicE-Cad/CTF2. Similarly, ionomycin decreased the cytoskeletal association(Triton X-100-insoluble fraction) of the CAJ components β-catenin andα-catenin with a concomitant significant increase in their solublecytosolic levels. These data indicate that ionomycin induces atime-dependent disassembly of the E-cadherin/catenin cytoskeletalcomplex resulting in increased production of the PS1/γ-secretasefragment E-Cad/CTF2 and in the solubilization of cytoskeletal β- andα-catenins. L-685,458 blocked the ionomycin-induced metabolism ofcytoskeletal E-Cad/CTF1 and partially inhibited degradation of the fulllength E-cadherin while it abolished production of soluble cytosolicE-Cad/CTF2, suggesting that L-685,458 inhibits the γ-secretase-likecleavage of both cytoskeletal full-length E-cadherin and E-Cad/CTF1. Inaddition, L-685,458 delayed the ionomycin-induced decrease ofcytoskeletal β-catenin and α-catenin and inhibited their release to thesoluble cytosol. These data show that the γ-secretase-like ε-cleavage ofE-cadherin promotes dissociation of the CAJ components from thecytoskeleton and their release to the soluble cytosol.

The PS1/γ-secretase role in the disassembly of CAJ was further examinedusing laser scanning confocal microscopy (LSCM). A431 cells werepre-incubated for 30 minutes in the absence or presence of L-685,458 andthen treated for 45 minutes with ionomycin. Following theionomycin-induced cell-cell dissociation, the distribution of PS1,E-cadherin, β-catenin and α-catenin was analyzed by LSCM using theconstant detector setting. Cells were double-labeled with eitheranti-PS2/NTF antibody R222 and anti-cytoplasmic E-cadherin antibody C36,or with anti-ectodomain E-cadherin antibody H108 and anti-β-cateninantibody. Cells were also labeled for α-catenin. PS1, cytoplasmic andectodomain sequences of E-cadherin, β-catenin and α-catenin concentratedat cell-cell contacts in confluent A431 cells. Ionomycin treatmentdisrupted cell-cell adhesion and decreased plasma membrane staining ofall epitopes. PS1, cytoplasmic E-cadherin, β-catenin and α-cateninstaining became more diffuse throughout the cytoplasm. In contrast,cadherin ectodomain staining was barely detectable suggesting asignificant reduction of the cellular levels of this epitope inagreement with the ionomycin-induced cleavage and secretion ofE-cadherin ectodomain.

Pre-incubation with L-685,458 significantly delayed loss of cell surfacestaining of all epitopes. Ectodomain E-cadherin staining was alsopartially preserved at the cell surface and at cell-cell contacts inagreement with the foregoing data that L-685,458 inhibits theionomycin-induced metabolism of cytoskeletal full-length E-cadherin.However, two-color immunofluorescence of L-685,458-treated cellsrevealed a cell population containing β-catenin but no ectodomainE-cadherin at the cell surface suggesting that these representjunctional complexes of β-catenin with E-Cad/CTF1. In L-685,458-treatedcultures, there was a complete junctional overlap between staining ofcyoplasmic E-cadherin and PS1 suggesting that cell surface PS1 remainsbound to both full-length E-cadherin and E-Cad/CTF1. This observationconcurs with the foregoing biochemical data showing that PS1 binds bothfull-length E-cadherin and E-Cad/CTF1.

Example 5 A Cadherin Mutant Unable to Bind PS1 is not Cleaved by thePS1/γ-Secretase Activity

To examine whether PS1 binding to E-cadherin is necessary for thePS1/γsecretase cleavage, E-cadherin mutant GGG759-761AAA was used(Thoreson et al., (2000) J. Cell Biol. 148:189-202). E-cadherin-negativeA431D cells were stably transfected either with WT E-cadherin or withthe E-cadherin mutant. Extracts from the transfected cells wereimmunoprecipitated with anti-PS1/NTF antibody R222, and theimmunoprecipitates were probed with anti-E-cadherin antibody C36 oranti-PS1/CTF antibody 33B10. In contrast to WT protein, mutantE-cadherin failed to bind PS1 consistent with reports that E-cadherinsequence 760-771 (corresponding to 604-615 residues of mature processedE-cadherin) is necessary for PS1/E-cadherin binding (Baki et al.,(2001)).

A431D cells stably transfected with WT E-cadherin or with the E-cadherinmutant were incubated in the absence or presence of ionomycin for 45minutes and RIPA extracts were probed on Western blots with C36antibody. Cytosolic fractions were probed with antibodies againstE-cadherin (C36), β-catenin or α-catenin. Upon ionomycin treatment,cells expressing WT E-cadherin showed a significant increase in solubleE-Cad/CTF2, β-catenin and α-catenin. In contrast, cells expressingmutant E-cadherin showed no ionomycin-induced increase in soluble β- orα-catenin. Furthermore, no soluble E-Cad/CTF2 was detected in mutanttransfectants either in the presence or absence of ionomycin. Thus, PS1binding to E-cadherin is required for the PS1/γ-secretase cleavage ofE-cadherin and for the release of E-Cad/CTF2, β-catenin and α-catenin tothe soluble cytosol.

Example 6 Overexpression of E-Cadherin Reduces Amyloid-β Production

L cell cultures were transfected with increasing amounts of humanE-cadherin. Amyloid-β (1-40) and amyloid-β (142) were measured by ELISA.FIG. 2 is a dose-response graph demonstrating that the production ofamyloid-β (1-40) decreases with increasing expression of E-cadherin.FIG. 3 is a bar graph depicting the production of amyloid-β (1-42) incultures of control (L) and E-cadherin-transfected (EL) cells. FIG. 4Ais a bar graph depicting the production of amyloid-β (1-40) in culturesof control (L) and E-cadherin-transfected (EL) cells. These resultsdemonstrate that overexpression of E-cadherin significantly reducesamyloid-β production.

CHO cells expressing Swedish APP mutant were transiently transfectedwith vector cDNA (vector), full length E-cadherin (E-cadherin), a cDNAconstruct encoding the transmembrane and ectodomain sequence ofE-cadherin (EC0) or a cDNA expressing the entire cytoplasmic sequence ofE-cadherin (CTF2). Secreted Aβ₄₀ and Aβ₄₂ were determined by ELISA.Results are shown in FIG. 4B. Each value is a mean±SD from three assayseach performed in duplicate. Expression of E-cadherin and CTF-2 but notEC0 significantly inhibited Aβ₄₀ and Aβ₄₂ (*p<0.001, **p<0.0001, ttest), suggesting that cytoplasmic sequences of E-cadherin areimplicated in the inhibition of Aβ. Expression of transfected proteinsin swCHO cells was monitored by Western blotting.

Example 7 FAD-PS1 Mutations Inhibit the γ-Secretase-Like ε-Cleavage ofN-Cadherin

HEK293 cells were stably transfected with wild-type or mutant (D2574A,ΔE9, A246E, E280A or M146L) PS1. Membrane preparations were incubatedand then analyzed by Western blots with either anti-cytoplasmicN-cadherin (FIGS. 5A and B, upper panels) or anti-PS1/N-terminalfragment (PS1/NTF) (FIGS. 5A and B, lower panels) antibodies. Theresults are shown in FIGS. 5A and 5B. Ncad/CTF1 and Ncad/CTF2 indicateC-terminal fragments of N-cadherin. Ncad/CTF1 is produced by ametalloproteinase activity, while Ncad/CTF2 is an ε-cleavage product. Inthe lanes numbered 7 in FIG. 5B, the cell line overexpressing wild-typePS1 was incubated in the absence (−) or presence (+) of the selectiveγ-secretase inhibitor, L-685,458.

As demonstrated in FIGS. 5A and 5B, compared to cells transfected withwild-type PS1, the production of Ncad/CTF2 was inhibited in cellstransfected with the PS1 mutants, and in cells transfected withwild-type PS1 and incubated with a γ-secretase inhibitor. Production ofNcad/CTF1 was comparable in cells transfected by wild-type and mutantPS1. These results demonstrate that PS1 mutations inhibit theγ-secretase-like processing of N-cadherin.

Membrane preparations were obtained from embryonic fibroblasts of PS1wild-type and PS1 P264L homozygous knock-in mice Siman et al. (2000) J.Neurosci. 20:8717-26. The preparations were analyzed by Western blotswith either anti-cytoplasmic N-cadherin (FIG. 6, upper three panels) oranti-PS1/C-terminal fragment (FIG. 6, lower panel) antibodies. Asdemonstrated in FIG. 6, ε-cleavage of N-cadherin was inhibited in thePS1 P264L knock-in mouse fibroblasts, but not in fibroblasts from PS1wild-type mice. Production of the metalloproteinase cleavage product,Ncad/CTF1, was not affected.

Example 8 Materials and Methods

The following materials and methods were used in the subsequentexamples.

Materials and antibodies. L-685,458 was obtained from Calbiochem, GM6001from Chemicon, D(−)-2-amino-5-phosphonovalerate (D-APV), L-Glutamate andN-methyl-D-aspartic acid (NMDA) were from Sigma. Rabbit polyclonalantiserum R222 against PS1/NTF residues 2-12 and mouse monoclonalantibody 33B10 specific for PS1/CTF residues 331-350 were prepared asdescribed by Georgakopoulos et al. (1999) Mo. Cell 4: 893-902.Anti-N-cadherin (clone C32) and anti-E-cadherin (C36) monoclonalantibodies were obtained from BD Transduction Laboratories.Anti-b-tubulin, anti-CBP (A-22) and anti-c-fos polyclonal antibodieswere from Santa Cruz Biotechnology. Antibodies against phosphorylatedCREB at Ser133 (1B6) was from Cell Signaling Technology.

Cell lines, cell culture and transfections. N2a, HEK293, L cells weregrown in Dulbecco's Modified Eagle's Medium (DMEM) plus 10% fetal bovineserum, penicillin and streptomycin in 5% CO₂ at 37° C. Embryonicfibroblasts from PS1 P264L-homozygous knock-in mice were prepared asdescribed by Siman et al. (2001) J. Neurosci. 20: 8717-8726. HEK293 celllines stably transfected either with WT or mutant PS1 cDNAs cloned intopC1-neo expression vector were grown in the presence of G418. L cellswere transfected with chicken cytoplasmic domain of N-cadherin(N-Cad/CTF2) inserted into vector pECE (pN-Cad/CTF2) or with full lengthmouse CBP inserted into vector pRc/RSV.

Immunoprecipitations (IPs), immunoblotting and immunofluorescence. IPswere performed as described by Marambaud et al. (2002) EMBO J. 21:1948-1956 in 4° C. HEPES buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 1×Complete protease inhibitor cocktail, Roche) containing 0.5% Nonidet P40(NP40). For western blot (WB) analysis, cells were solubilized in 4° C.RIPA buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.5%sodium deoxycholate, 1× Complete protease inhibitor cocktail) asdescribed by Marambaud et al. (2002). Immunofluorescence was performedas described by Georgakopoulos et al. (1999) with the followingmodifications: to visualize nuclei, cells were treated for 5 minuteswith 4′,6-diamidino-2-phenylndole (DAPI, 1:5000; Sigma). Fluorescencemicroscopy and digital image acquisition were carried out using anAxioskop2 microscope (Zeizz).

Mouse embryo preparation. Mouse PS1+/+, +/− or −/− embryos or mouseembryonic brains were collected at E18.5 and solubilized in RIPA bufferas described by Baki et al. (2001) Proc. Natl. Acad. Sci. USA 98:2381-2386. Fifty micrograms of extract were analyzed on WBs.

Primary, neuronal cultures. Mixed hippocampal and cortical neuronalcultures were prepared from E18 mouse or rat brains as described byBanker and Goslin (1991) Culturing Nerves Cells, London, MIT Press.Neurons were maintained 10 days in vitro in glial conditioned MEMcontaining 2% glucose and N2 supplements as described by Bottenstein andSato (1979) Proc. Natl. Acad. Sci. USA 76: 514-517, and then werestimulated for 15 minutes with Mg2+-free Hanks' balanced salt solution(HBSS, Sigma) containing KCl (50 mM), L-Glutamate (50 mM) or NMDA (50mM). D-APV (100 mM) was applied 15 minutes before stimulation.

In vitro γ-secretase-like ε-cleavage assay. Cells were washed in PBS,resuspended in 1 ml of 4° C. hypotonic buffer (10 mM MOPS, pH 7.0, 10 mMKCl) and homogenized on ice. A post-nuclear supernatant was prepared bycentrifugation at 1000×g for 15 minutes at 4° C. Crude membranes wereisolated from the post-nuclear supernatant by centrifugation at 16,000×gfor 40 minutes at 4° C. The membranes were then resuspended in 25 ml ofassay buffer (150 mM sodium citrate, pH 6.4, 1× Complete proteaseinhibitor cocktail), and incubated at 37° C. for 2 hours. Samples wereeither analyzed directly by WB or separated into pellet (P100) andsupernatant (S100) fractions by ultracentrifugation for 1 hour at100,000×g at 4° C. The S100 and P100 fractions were then analyzed by WB.

Transactivation assays. CRE-dependent transactivation was measured inthe absence or presence of overexpression of PKA (pFC-PKA, Stratagene)by cotransfection with CRE-luciferase reporter plasmid (pCRE-Luc,Stratagene) and pSV-β-galactosidase vector (Promega) to evaluatetransfection efficiency. CHOP-mediated transactivation was measured bycotransfection with the Gal4-fusion trans-activator plasmids (pFA-CHOPand pFR-Luc, Stratagene). 30 hours after transfection, luciferase andβ-galactosidase activities were determined following manufacturer'sinstructions using a TD-20/20 luminometer (Turner Designs) and a DU-64spectrophotometer (Beckman). Values were normalized to β-galactosidaseactivity and protein concentration.

Subcellular fractionation. Transfected cells were washed in PBS, placedinto 1 ml of buffer A (20 mM Tris-HCl, pH 7.5, 0.25 M sucrose, 10 mMEGTA, 2 mM EDTA, Ix Complete protease inhibitor cocktail), and thenpassed through a 27 gauge needle 10 times. Obtained lysates werecentrifuged at 500×g for 10 minutes and pellets (nuclear fraction) werewashed twice with 1 ml of buffer A containing 0.1% Triton X-100 andsolubilized by sonication in RIPA buffer. Supernatants were centrifugedat 120,000×g for 45 minutes at 4° C. to separate cytosolic and crudemembrane fractions. A 50 μg protein aliquot from the nuclear andcytosolic fractions was analyzed on WBs.

Reverse Transcriptase (RT)-PCR Analysis. Total RNA was extracted fromcells using a RNeasy Mini Kit (Qiagen) according to instructions by themanufacturer. Analysis of gene expression was performed usingsemiquantitative RT-PCR. The following primers were used:5′-GGGTTTCAACGCCGACTACG-3′ (SEQ ID NO: 13) and5′-CAGCTTGGGAAGGAGTCAGC-3′ (SEQ ID NO: 14) for c-fos;5′-TGTCGTGGAGTCTACTGG-3′ (SEQ ID NO: 15) and 5′-CAGCATCAAAGGTGGAGG-3′(SEQ ID NO: 16) for gapdh.

Electrophoresis mobility shift assay (EMSA). Nuclear extract fromCBP-transfected L cells were prepared using the extraction kit N-XTRACT(Sigma) according to manufacturer's instructions. Ten microgram ofnuclear extract was used for EMSA performed according to manufacturer'sinstructions using biotinylated double-stranded oligonucleotide probescontaining DNA-binding motifs for CREB or MEF-1 (Panomics). Probes areas follow: CREB, 5′-AGAGATTGCCTGACGTCAGAGAGCTAG-3′ (SEQ ID NO: 17);MEF-1,5′-GATCCCCCCAACACCTGCTGCCTGA-3′ (SEQ ID NO: 18). Reaction productswere separated on 6% polyacrylamide gels in cold TBE buffer (50 mMTris-HCl, 45 mM Boric acid, 0.5 mM EDTA), transferred to nylon membranes(Immobilon-Ny+, Millipore) and bound probes were immobilized for 30minutes at 85° C. and visualized by chemiluminescence usingstreptavidin-HRP conjugate. For antibody supershift analysis, nuclearextract was incubated for 3 hours at 4° C. with 2 ml of 1 B6 antibodyprior to probe addition. Where indicated, nuclear extract (20 mg) wasimmunoprecipitated with A-22 antibody (1 ml) and protein A-agarose inEBC buffer (50 mM Tris, pH 8, 120 mM NaCl, 0.5% NP40, 1× Completeprotease inhibitor cocktail). Immunoprecipitates were washed twice withEBC buffer and twice with buffer B (20 mM HEPES, pH 7.5, 50 mM KCl, 10mM MgCl2, 10% glycerol, 0.5 mM DTT). Immunoprecipitates were then elutedwith 0.8% sodium deoxycholate in buffer B. The supernatants were made1.2% NP40 and used for EMSA.

Example 9 PS1-Mediated γ-secretase-Like Activity Cleaves N-cadherin

Extracts from PS1+/+ or PS1−/− mouse embryos were probed on WBs witheither anti-cytoplasmic N-cadherin antibody (FIG. 7A, upper panel) oranti-PS I/CTF antibody 33B10 (FIG. 7A, lower panel). Extracts fromPS1+/+ or PS1−/− fibroblast cultures were incubated for 16 hours in theabsence or presence of DMSO, L-685,458 (0.5 μM) and probed on WBs witheither C32 or 33B10 antibodies.

PS1 knockout (PS1−/−) mouse embryos accumulated a 40 kDa peptide (termedN-Cad/CTF1) detected with antibodies against the cytoplasmic sequence ofN-cadherin. This peptide was hardly detectable in wild type (WT) mouseembryos although all embryos contained similar amounts of full lengthN-cadherin (FIG. 7A). A similar peptide accumulates in PS1−/−fibroblasts or in WT fibroblasts treated with the γ-secretase inhibitorL-685,458 (Li et al. (2000) Nature 405:689-694,) but not in control WTfibroblasts. All cultures contained similar amounts of full lengthN-cadherin (FIG. 7B, lanes 1-4). Matrix metalloproteinase (MMP)inhibitor GM6001 decreased N-Cad/CTF1 and increased full lengthN-cadherin (FIG. 7B, lanes 5 and 6). Together, these data indicate thatN-Cad/CTF1 derives from full length N-cadherin through a MMP cleavageand is subsequently processed by a PS1-dependent γ-secretase-likeactivity. The corresponding peptide derived through the MMP cleavage ofE-cadherin, a protein highly homologous to N-cadherin, is demonstratedin Example 2 hereinabove.

To detect the product of the PS1-dependent cleavage of N-cadherin, an invitro assay that uses incubation of membrane fractions to detect the ICDproduct of the E-cleavage of APP was used. (Gu et al. (2001) J. Biol.Chem. 276:35235-35238; McLendon et al. (2000) FASEB J. 14:2383-2386).

Membranes from PS1−/− (FIG. 7C, lane 1) or PS1+/+ (FIG. 7C, lanes 2-4)fibroblasts or from N2a cell cultures (FIG. 7C, lanes 5-6) incubated for16 hours in the absence or presence of DMSO or L-685,458 (0.5 mM), wereincubated at 37° C. for 2 hours, separated by centrifugation in a pellet(P100, FIG. 7C, upper panels) and a soluble (S100, FIG. 7C, lowerpanels) fractions and then probed on WBs with C32 antibody. Membranesfrom HEK293 cell cultures stably transfected with vector alone (FIG. 7D,lanes 1 and 2), WT-PS1 (FIG. 7D, lanes 3-5), or D257A-PS1 (FIG. 7D,lanes 6 and 7) were incubated at 37° C. for 2 hours and then probed witheither C32 (FIG. 7D, upper panel), anti-PS1/NTF (FIG. 7D, middle panel)or anti-PS1/CTF (FIG. 7D, lower panel) antibodies. Membranes fromWT-PS1-transfected clone #7 were incubated in the absence (−) orpresence (+) of L-685,458 (FIG. 7D, lanes 4 and 5).

A soluble 35 kDa peptide, termed N-Cad/CTF2, containing the cytoplasmicsequence of N-cadherin was detected in this assay using membranefractions from either mouse fibroblasts or from mouse neuroblastoma cellline N2a (FIG. 7C). N-Cad/CTF2 is greatly reduced in the absence of thePS1 gene and is completely absent from cultures treated with γ-secretaseinhibitor L-685,458 (FIG. 7C). The residual N-Cad/CTF2 detected inPS1−/− cells is probably due to activity of PS2, a PS1 homologue(Levy-Lahad et al. (1995) Science 269:970-973; Rogaev et al. (1995)Nature 376:775-778). Overexpression of PS1 in HEK293 cells resulted in asignificant stimulation of N-Cad/CTF2 production compared to membranesfrom vector-transfected cells (FIG. 7D, lanes 1-4). In contrast, noN-Cad/CTF2 was detected using membranes prepared either from cellsoverexpressing the γ-secretase dominant negative PS1 mutant D257A (Wolfeet al. (1999) Nature 398:513-517) (FIG. 7D, lanes 6 and 7) or from cellstreated with γ-secretase inhibitor L-685,458 (FIG. 7D, lanes 4 and 5).Together with the apparent SDS-PAGE molecular mass of N-Cad/CTF2, thesedata indicate that N-cadherin undergoes a ε-cleavage by thePS1/γ-secretase system to release an ICD peptide of N-cadherin. Example3 hereinabove shows that E-cadherin is processed by a similar mechanismto release E-Cad/CTF2.

Example 10 NMDA Receptor Agonists or Membrane Depolarization Stimulatesthe PS1/ε-Cleavage of N-Cadherin

N-cadherin and PS1 are both expressed in neurons and in brain tissuethey are found in the same complex (Georgakopoulos et al. (1999) Mol.Cell 4:893-902). N-cadherin is a synaptic component that undergoesstructural changes following stimulation of the N-methyl-D-aspartate(NMDA) receptor (Tanaka et al. (2000) Neuron 25:93-107). PS1 has alsobeen localized at the synapse (Georgakopoulos et al. (1999);Ribaut-Barassin et al. (2000) Synapse 35:96-110). To examine whether thePS1/γ-secretase system is involved in the processing of neuronalN-cadherin, membranes from a culture of primary neurons prepared frombrains of WT and PS1−/− mouse embryos were tested. Membranes preparedfrom PS1−/− (FIG. 8A, lane 1) PS1+/+ (FIG. 8A, lane 2) or PS1+/− (FIG.8A, lanes 3 and 4) mouse brain neuronal cultures were incubated at 37°C. for 2 hours. PS1+/− neurons were incubated in the absence (−) orpresence (+) of L-685,458 (0.5 μM). Following incubation samples wereprobed with C32 (FIG. 8A, upper panel) or R222 (FIG. 8A, lower panel).

FIG. 8A (lanes 1 and 2) shows that N-cadherin ICD fragment N-Cad/CTF2 isdetected in membranes from WT cultures but not in membranes from PS1−/−cultures. Furthermore, N-Cad/CTF2 peptide is inhibited by γ-secretaseinhibitor L-685,458 (FIG. 8A, lanes 3 and 4).

Ionomycin, an agent that stimulates calcium influx, induces thePS1/ε-cleavage of E-cadherin as demonstrated in Example 4 hereinabove.Treatment of rat primary neuronal cultures with this agent increasedN-Cad/CTF2 suggesting that calcium stimulates the PS1/E-cleavage ofN-cadherin. To determine whether more specific treatments that stimulatecalcium influx through channels would also affect the E-cleavage ofN-cadherin, the following experiment was performed. Rat brain neuronswere pre-incubated in the absence (−) or presence (+) of D-APV and thenstimulated with either KCl, L-glutamate (L-gly) or NMDA. Membranes fromthese neurons were used in in vitro assays for production of N-Cad/CTF2.FIG. 8B (lanes 1 and 3) shows that treatment of rat primary neuronalcultures with 50 mM KCl stimulated production of peptide N-Cad/CTF2.

Since high K+ depolarizes neuronal membranes resulting inneurotransmitter release at synaptic endings (Buchs and Muller (1996)Proc Nat. Acad. Sci USA 93:8040-8045), it was determined whetherblocking specific postsynaptic receptors would inhibit the increase inN-Cad/CTF2. FIG. 8B (lanes 14) shows that treatment of neurons withD(−)-2-amino-5-phosphonovalerate (D-APV), a specific antagonist of theNMDA receptor, inhibited the K+-induced increase in N-Cad/CTF2,indicating involvement of this receptor in the PS1/ε-cleavage ofN-cadherin. Direct stimulation of the NMDA receptor using agonistL-glutamate also induced an increase in N-Cad/CTF2 and this increase wasblocked by D-APV (FIG. 8B, lanes 1, 5 and 6). Application of NMDA hadsimilar results (FIG. 8B, lanes 1 and 7). These data indicate that thePS1/ε-cleavage of neuronal N-cadherin is stimulated by membranedepolarization and activation of the NMDA receptor.

Example 11 N-Cad/CTF2 Down-Regulates CREB-Mediated Transcription

Stimulation of synaptic NMDA receptor leads to phosphorylation oftranscription factor CREB that then recruits transcription co-activatorCBP, a process ultimately leading to activation of CREB-mediated geneexpression (Impey and Goodman (2001) Sci STKE, PE1). The functionalconsequences of increased N-Cad/CTF2 production were examined todetermine whether this peptide affects CRE-dependent transactivation, aprocess that relies on endogenous CBP and CREB. L cells were transfectedwith increasing amount of pN-Cad/CTF2. CRE-dependent transactivation wasmeasured in the absence (−) or presence (+) of β-transfected PKA(pFC-PKA) using CRE-luciferase reporter plasmid (pCRE-Luc) andpSV-β-galactosidase vector. CHOP-mediated transactivation was measuredby β-transfection with the Gal4-fusion trans-activator plasmids(pFA-CHOP and pFR-Luc) and pSV-β-galactosidase vector. Transfected cellswere processed for luciferase activity. Results are shown in FIG. 9A.

HEK293 cultures stably transfected either with vector or WT-PS1 wereco-transfected with pFC-PKA, pCRE-Luc, and pSV-β-galactosidase in thepresence or absence of L-685,458 and then processed for CRE-dependentluciferase activity. Data in FIGS. 9A and B are the mean+/−s.e. of 4experiments normalized to β-galactosidase activity and proteinconcentration. *, P<0.05; ***, P<0.001 (Student's t test).

Semi-quantitative RT-PCR was used to determine relative levels of c-fosor gapdh mRNAs from L cells transfected either with vector (vector) orwith 0.5 mg of pN-Cad/CTF2 (N-Cad/CTF2). The graph in FIG. 9C representsthe relative abundance of c-fos-specific PCR products followingquantitation of the mRNA signals shown in the upper panel.

Extracts from L cells transfected with increasing amount of pN-Cad/CTF2were probed on WBs with anti-c-fos (FIG. 9D, upper panel),anti-β-tubulin (FIG. 9D, middle panel) or C32 (FIG. 9D, lower panel)antibodies. Bars in lower panel represent the relative amounts of c-fosprotein detected on WBs shown in the upper panel.

FIG. 9A shows that transfection of N-cadherin-negative L cells withN-Cad/CTF2 inhibits both constitutive and PKA-stimulated CRE-dependenttransactivation in a dose-dependent manner. Transactivation byCHOP-response elements was not affected by N-Cad/CTF2 (FIG. 9A)indicating that this peptide specifically represses CRE-dependenttransactivation. Since PS1 overexpression increases N-Cad/CTF2production (FIG. 7D), these data predict that PS1 transfection shoulddecrease CRE-dependent transactivation. Indeed, FIG. 9B shows that thisis the case. PS1 overexpression decreases CRE-dependent transactivationand this decrease is reversed by γ-secretase inhibitor L-685,458 whichinhibits N-Cad/CTF2 production (FIG. 7D) further supporting thesuggestion that the PS1/ε-cleavage product N-Cad/CTF2 suppressesCREB-mediated transcription. Similarly, overexpression of the dominantnegative mutant D257A that inhibits production of N-Cad/CTF2 (FIG. 7D)also stimulates CRE-dependent transactivation (FIG. 12D). These datashow an inverse correlation between N-Cad/CTF2 production andCRE-dependent transactivation, indicating that N-Cad/CTF2 acts as arepressor of CREB-mediated transcription.

To confirm that N-Cad/CTF2 suppresses CREB-dependent transcription ofendogenous genes, the expression of c-fos mRNA and protein change inresponse to this peptide was assessed. The c-fos promoter contains CREsand CREB is a strong regulator of the transcription of this gene (Ahn etal. (1998) Mol. Cell. Biol. 18:967-977). FIGS. 9C and 9D show thatoverexpression of N-Cad/CTF2 markedly reduces the amounts of cellularc-fos mRNA and c-fos protein but has no effect on the expression ofgapdh mRNA or on the levels of β-tubulin suggesting that N-Cad/CTF2specifically inhibits c-fos expression. To determine whether conditionsthat inhibit both PS1 activity and N-Cad/CTF2 production, like L-685,458treatment or absence of PS1, would affect CREB-mediated transcription,the following experiment was performed.

N2a cells co-transfected with plasmids pCRE-luc, pFC-PKA, andpSV-β-galactosidase were incubated for 16 hours with DMSO or withL-685,458 (0.5 mM) and CRE-dependent transactivation was measured asdescribed above. Data in FIG. 10A are the mean+/−s.e. of 3 experimentsnormalized to β-galactosidase activity and protein concentration. *,P<0.05 (Student's t test). Total RNA isolated from N2a cells treatedeither with DMSO or with L-685,458, was analyzed by semiquantitativeRT-PCR using specific primers for c-fos and gapdh. The graph in FIG. 10Brepresents the relative abundance of c-fos-specific PCR productsfollowing quantitation of the mRNA signals shown in the upper panel.Extract from N2a cells treated either with DMSO or with L-685,458, wereprobed on WBs with anti-c-fos or anti-β-tubulin antibodies. Results areshown in FIG. 10C. Extract from PS1+/+ or PS1−/− mouse fibroblasts wereprobed on WBs with either anti-c-fos or anti-β-tubulin antibodies.Results are shown in FIG. 10D. Extract from PS1+/− or PS1−/− mouseembryonic brains were probed on WBs with anti-c-fos (FIG. 10E, upperpanel), anti-b-tubulin (FIG. 10E, middle panel), or anti-PS1/CTF (FIG.10E, lower panel) antibodies.

L-685,458 treatment of N2a cells increased CRE-dependent transactivation(FIG. 10A) and stimulated c-fos mRNA (FIG. 10B) and c-fos protein (FIG.10C) beyond the levels observed in untreated controls, whereas gapdhmRNA and β-tubulin remained unchanged (FIGS. 10B and 10C). Furthermore,FIGS. 10D and 10E show that absence of PS1 results in abnormally highlevels of c-fos protein in both fibroblast cultures and mouse brainwhile levels of β-tubulin remain unchanged. Together, these data showthat downregulation of PS1 activity results in a decreased production ofN-Cad/CTF2 and in a specific overexpression of c-fos indicating that PS1activity is needed for maintaining normal expression of this gene.

Example 12 N-Cad/CTF2 Binds and Sequesters CBP in the Cytoplasm

To determine how N-Cad/CTF2 affects CREB-mediated transcription, thefollowing experiments were performed. L cells transfected either withvector (vector) or pN-Cad/CTF2 (N-Cad/CTF2) plasmids were fractionatedinto nuclear and cytosolic fractions, and probed on WBs with anti-CBP(FIG. 11A, panel a), anti-N-cadherin (FIG. 11A, panel b), anti-β-tubulin(FIG. 11A, panel c) or anti-phosphorylated CREB (P-CREB; FIG. 11A, paneld) antibodies. Extract from pN-Cad/CTF2-transfected L cells wasimmunoprecipitated either with non-immune serum (NI) or with antibodiesagainst CBP (CBP), β-tubulin (β-Tub), E-cadherin (C36) or N-cadherin(C32). Obtained IPs were probed with anti-CBP (FIG. 11B, upper panel),anti-N-cadherin (FIG. 11B, middle panel) or anti-β-tubulin (FIG. 11B,lower panel) antibodies. For reference, cell lysate was also probed(FIG. 11B, first lane). L cells transiently transfected with pN-Cad/CTF2were analyzed by immunofluorescence staining. Cells were triple-labeledwith DAPI (blue), anti-CBP (red) and anti-N-cadherin (C32; green)antibodies. Nuclear extract from pN-Cad/CTF2-transfected L cells wereincubated with biotinylated double-stranded DNA probes containingbinding sequence for either CREB (FIG. 11C, left panel) or MEF-1 (FIG.11C, right panel). The lanes in FIG. 11C are as follows: Lanes 1 and 9:probes alone; Lanes 2 and 10: probe plus extract; Lanes 3 and 11:samples as in lanes 2 and 10 plus a 60-fold excess of unlabelled (cold)probes used as competitors; Lane 4: incubation was carried out in thepresence of anti-phosphorylated CREB-Ser133 antibody 1B6; Lane 5:nuclear extract was immunoprecipitated with anti-CBP antibody A-22 andthe obtained IPs were eluted with sodium deoxycholate, incubated withCREB probe and loaded on the gel; Lanes 6-8 and 12-14: nuclear extractfrom cells transfected without (lanes 6 and 12) or with 0.2 μg (lanes 7and 13) and 0.5 μg (lanes 8 and 14) of pN-Cad/CTF2 were incubated eitherwith CREB (lanes 6-8) or MEF-1 (lanes 12-14) probes and then loaded onthe gel. F indicates free probes. Shifted probes (S) were competed outwith unlabeled probes (lanes 3 and 11). CREB probe was supershifted (Ss)with antibody against phosphorylated CREB (lane 4) and the shifted CREBprobe was also obtained after immunoprecipitation of nuclear extractswith CBP antibody (lane 5), indicating that the DNA-bound complexcontains both CREB and CBP.

CREB-mediated transcription is stimulated by phosphorylation ofCREB-Ser133 and CBP recruitment to transcription initiation complexesformed at CRE-containing promoters (Vo and Goodman (2001) J. Biol. Chem.276:13505-13508. However, any change of CREB phosphorylation in responseto N-Cad/CTF2 overexpression was not detected. (FIG. 11A, panel d). Theability of this peptide to affect CREB-mediated transcription bylimiting the availability of CBP was assessed. Cell fractionation andbiochemical studies showed that as expected (Chrivia et al. (1993)Nature 365:855-854), in vector-transfected cells, CBP was found only inthe nuclear fraction where phosphorylated CREB is also found (FIG. 11A,lanes 1 and 3, panels a and d). Following transfection with N-Cad/CTF2,however, nuclear CBP decreased with a concomitant increase in cytosolicCBP, indicating that in the presence of N-Cad/CTF2, CBP translocates tothe cytoplasm where N-Cad/CTF2 is localized (FIG. 11A, panels a and b).In contrast to CBP, N-Cad/CTF2 had no effects on the localization ofphosphorylated CREB, which remained in the nucleus (FIG. 11A, panel d).Additional experiments showed that in N-Cad/CTF2-transfected cells, CBPco-immunoprecipitates with N-Cad/CTF2 but not with β-tubulin. Thereverse is also true, N-Cad/CTF2 co-immunoprecipitates with CBP (FIG.11B). These data indicate that the two proteins form a complex in thecytoplasm. In agreement with the biochemical and immunoprecipitationstudies, two-color immunofluorescence staining showed that compared tonon-transfected controls which display no CBP staining in the cytoplasm,cells transfected with N-Cad/CTF2 show a clear cytoplasmic staining forboth N-Cad/CTF2 and CBP. In contrast to CBP, CREB staining remainednuclear and showed no co-localization with N-Cad/CTF2 staining in thetransfected cells. Electrophoretic mobility shift assays (EMSA) wereused to examine the effects of N-Cad/CTF2 on formation of a CBP/CREBcomplex on its cognate CRE motif. FIG. 11C (lanes 6-8) shows thatnuclear extract from N-Cad/CTF2-transfected cells are defective in theirability to promote formation of a CBP/CREB complex on CRE-containing DNAtemplates. In contrast, N-Cad/CTF2 transfection had no effect on theability of this nuclear extract to promote complex formation betweentranscription factor MEF-1 and a DNA probe containing MEF-1-bindingelements (FIG. 11C, lanes 12-14). Together, these data show thatN-Cad/CTF2 binds with CBP and sequesters it to the cytoplasm thuslimiting its availability for formation of nuclear CREB/CBP/DNAcomplexes.

Example 13 PS1 FAD Mutants do not Stimulate N-Cad/CTF2 Production andare Unable to Suppress CREB-Mediated Transcription

To examine the effects of PS1 FAD mutations on the ε-cleavage ofN-cadherin, the amounts of N-Cad/CTF2 produced in the in vitro assayusing membranes from HEK293 cells over-expressing comparable levels ofeither WT or PS1 FAD mutants were measured. Membranes from twoindependent clones of HEK293 cells each stably transfected with vectoralone (FIG. 12A, #1 and 6), WT-PS1 (FIG. 12A, #3 and 7), or with PS1mutants M146L (FIG. 12A, #8 and 14), A246E (FIG. 12A, #1 and 7), E280A(FIG. 12A, #5 and 6), ΔE9 (FIG. 12A, #4 and 8), V82L (FIG. 12B, #1 and11), G384A (FIG. 12B, #3 and 4), E280G (FIG. 12B, #5 and 8) and Y115H(FIG. 12B, #8 and 9) were incubated in vitro and produced N-Cad/CTF2 wasdetected on WBs with C32 (FIGS. 12A and B, upper panels). Numberscorrespond to isolated clones. PS1 fragments were detected with R222(FIG. 12A, middle panel) or 33B10 (FIGS. 12A and B, lower panels). FIG.12C depicts combined densitometric quantitation of N-Cad/CTF2 using twoindependent clones of each mutant (FIGS. 12, A and B). N-Cad/CTF2 levelswere normalized to N-Cad/CTF2 produced from vector-transfected cells.Bars represent the mean+/−s.e. of three independent experiments usingtwo clones for each mutant. CRE-dependent transactivation was measuredusing two independent HEK293 cell stable clones of each mutant (FIGS.12, A and B). All clones were co-transfected with pFC-PKA, pCRE-Luc, andpSV-β-galactosidase. Luciferase activity data were normalized toβ-galactosidase activity and protein concentration. Results are shown inFIG. 12D. Bars represent the mean+/−s.e. of three experiments. *,P<0.05; **, P<0.005; ***, P<0.001 (Student's t test).

FIGS. 12A, B and C show that membranes from HEK293 cell culturesoverexpressing WT PS1 produce approximately seven times as muchN-Cad/CTF2 as the vector-transfected controls. In contrast, membranesfrom HEK293 cell cultures each overexpressing a PS1 FAD mutant carryingone of the missense mutations Y115H, M146L, A246E, E280A, E280G andG384A or the deletion mutation ΔE9, showed no increase in N-Cad/CTF2production indicating that these mutations are strong inhibitors of thePS1-dependent ε-cleavage of N-cadherin. PS1 FAD mutant V82L was the onlymutant that showed substantial E-cleavage although it was less activethan the WT PS1 (FIGS. 12, B and C).

That FAD mutations inhibit production of N-Cad/CTF2, a peptide thatbinds CBP and suppresses CREB-mediated transcription, predicts that PS1FAD mutants should be defective in their ability to suppressCRE-dependent transactivation. Indeed, FIG. 12D shows that althoughoverexpression of WT PS1 significantly suppressed CRE-dependenttransactivation, PS1 FAD mutants Y115H, M146L, A246E, E280A, E280G, ΔE9and G384A are unable to inhibit CRE transactivation. FAD mutants M146L,A246E, E280G and G384A as well as the γ-secretase dominant negativemutant D257A showed a clear dominant positive effect, increasingCRE-dependent transactivation over that of the vector control (FIG.12D). In agreement with its effects on N-Cad/CTF2 production,overexpression of FAD mutant PS1 V82L failed to increase significantlythe CRE-dependent transactivation compared to WT PS1 transfectedcontrols. This FAD mutant does not significantly increase production ofAβ42 peptide in cell cultures either (Murayama et al. (1999) Neuro Sci.Len. 265:61-63). The inverse correlation between the effects of PS1mutants on N-Cad/CTF2 production and CRE-dependent transactivation,observed in all cases, indicates that these mutants over-stimulatetranscription by inhibiting production of N-Cad/CTF2.

To investigate the consequences of FAD mutations in a physiologicallyrelevant system in the absence of PS1 overexpression, the ε-cleavage ofN-cadherin was examined in two independent embryonic fibroblast clonesderived from a gene-targeted (knock-in) mouse homozygous for the humanPS1 FAD mutation P264L (Siman et al. (2000) J. Neurosci. 20:8717-8726).Expression of the knock-in mutant PS1 allele is under the control of theendogenous PS1 gene. These models are distinct from overexpressingtransgenic models because the mutant gene is expressed at normal levels,similar to those observed in WT models (FIG. 13, panel d). As controls,two WT fibroblast cell lines independently derived from WT littermateswere used. Membranes from embryonic fibroblast cultures isolated eitherfrom PS1 P264L homozygous knock-in mice (KI-3 and KI-1) or from WTlittermates (WT-3 and WT-2) were processed for the generation ofN-Cad/CTF2. Following incubation, membranes were probed with C32 (FIG.13A, panels a-c) or 33B10 (FIG. 13A, panel d). Protein extract eitherfrom WT (WT-3 and WT-2) or from PS1 P264L knock-in (KI-3 and KI-1)fibroblasts were probed on WBs with anti-c-fos (FIG. 13B, upper panels)and anti-b-tubulin (FIG. 13B, lower panels) antibodies.

Production of N-Cad/CTF2 was sensitive to γ-secretase inhibitorL-685,458 (FIG. 13A, lanes 1 and 2). FIG. 13A (lanes 2-5) shows thatsamples from the knock-in fibroblast cell lines produced undetectableamounts of N-Cad/CTF2 compared to samples from the WT clones, indicatingthat this FAD mutation also inhibits the γ-cleavage of N-cadherin.Furthermore, the PS1 P264L knock-in cells contained significantly higherlevels of endogenous c-fos compared to WT controls, although β-tubulinlevels remained unchanged (FIG. 13B). Thus, the PS1 FAD mutation P264Linhibits N-Cad/CTF2 production and increases the cellular levels ofc-fos protein above those detected in normal controls indicating thatthis mutation induces a dysregulation of c-fos expression by inhibitingthe PS1/ε-secretase activity. FIG. 13C is a schematic representation ofthe effects of the PS1/e-cleavage of N-cadherin on CBP/CREB signaling.This cleavage releases N-cadherin ICD N-Cad/CTF2 that binds CBP andsequesters it to the cytoplasm thus downregulating CREB-mediatedtranscription. PS1 FAD mutations inhibit N-Cad/CTF2 production andstimulate CBP/CREB-dependent transcription. In neuronal cells, thec-cleavage is stimulated by NMDA receptor activation.

1. A peptide that binds to presenilin-1 and comprises the sequence EGGGE(SEQ ID NO: 5) wherein the peptide is from five to fifteen amino acidsin length.
 2. The peptide of claim 1 comprising a sequence selected fromthe group consisting of EGGGEEDQDFDL (SEQ ID NO: 1), EGGGEMDTTSYD (SEQID NO: 2), EGGGEEDQDYDLS (SEQ ID NO: 3) and EGGGEED (SEQ ID NO: 4).
 3. Acomposition comprising the peptide of claim
 1. 4. A method of inhibitingPresenilin-1-mediated γ-secretase activity comprising contacting a cellcapable of exhibiting such activity with the composition of claim 1.